Cryopreservation of plant cells

ABSTRACT

The present invention relates to methods for cryopreserving plant cells and to methods for recovering viable plant cells from long or short term cryopreservation. Plant cells to be cryopreserved can be grown in culture and pretreated with a solution containing an cryoprotective agent and a stabilizer. Pretreated cells are acclimated to a reduced temperature and loaded with a cryoprotective agent such as DMSO, propylene glycol or polyethylene glycol. Loaded cells are incubated with a vitrification solution which, for example, comprises a solution with a high concentration of the cryoprotective agent. Vitrified cells retain less than about 20% water content and can be frozen at cryopreservation temperatures for long periods of time without significantly altering the genotypic or phenotypic character of the cells. Plant cells may also be cryopreserved by lyophilizing cells prior to exposure to a vitrification solution. The combination of lyophilization and vitrification removes about 80% to about 95% of the plant cell&#39;s water. Cells can be successfully cryopreserved for long periods of time and viably recovered. The invention also relates to methods for the recovery of viable plant cells from cryopreservation. Cells are thawed to about room temperature and incubated in medium containing a cryoprotective agent and a stabilizer. The cryoprotective agent is removed and the cells successfully incubated and recovered in liquid or semi-solid growth medium. The invention also relates to the cryopreserved cells and to viable plant cells which have been recovered from long or short term cryopreservation.

BACKGROUND OF THE INVENTION

1. Field of the Invention

The present invention relates to methods for the cryopreservation ofplant cells and to methods for the recovery of plants cells which havebeen cryopreserved. The invention also relates to plants, viable plantcells and plant cells cultures which have been successfully recoveredfrom cryopreservation.

2. Description of the Background

Cryopreservation is based on the reduction and subsequent arrest ofmetabolic functions of biological material stored in ultra-lowtemperatures. Cryogenic preservation of plants and plant cells forextended periods without genetic change and the subsequent recovery ofnormal plant cells with unaltered characteristics and biosyntheticability has important implications in plant breeding, biomedicalresearch and genetic engineering. At the temperature of liquid nitrogen(-196° C.) almost all metabolic activities the cell ceases and cells canbe maintained in this suspended, but viable state for extended periods.

Plant cells are cryopreserved to avoid loss by contamination, tominimize genetic change in continuous cell lines, and to avoid aging andtransformation in finite cell lines. Traditional methods forpreservation of a desirable plant characteristic involve establishmentof colonies of plants in the field because many plants do not breed truefrom seeds. These field plant depositories demand large inputs of laborand land and incur high risks of loss due to weather, disease or otherhazards. An alternative to field colony is the establishment of an invitro collection of plant tissue under normal or limited growthconditions. For long-term storage, elimination of routine subculturingis desirable because of concerns with mutation, contamination, laborcost and risk of human error associated with tissue culture.

Most biological materials, including plants, cannot survive freezing andthawing from cryogenic temperatures without cryoprotective agents andprocedures. A number of cryopreservatives possess properties which canprotect a cell from the damaging effects of cryogenic freezing. Theessence of cryopreservation is to effect cell dehydration andconcentration of the cytosol in a controlled and minimally injuriousmanner so that ice crystallization in the cytosol is precluded orminimized during, for example, quenching in liquid nitrogen.

In conventional cryopreservation procedures, cell dehydration iseffected by freeze-induced concentration of the suspending medium.Deleterious effects of dehydration are mitigated by the presence ofcryoprotective agents. Specimens such as cells and organs areequilibrated in a solution containing a cryopreservation agent such asdimethylsulfoxide (DMSO) or ethylene glycol. The suspension is cooledand seeded with an ice crystal at a temperature slightly below itsfreezing point. The suspension is cooled again at an optimum rate to anintermediate sub-zero temperature such as between about -30° C. to about-40° C. and finally quenched in liquid nitrogen.

While routine cryogenic preservation of microorganisms, zygotes andanimals derived from zygotes is possible, the cryopreservation of plantcells is far from routine and often, different protocols for individualspecies of plants are necessary.

Taxus trees produces taxol, is a diterpenoid alkaloid originallyisolated from the bark of the Pacific yew, Taxus brevifolia (M. C. Waniet al., J. Am. Chem. Soc. 93:2325-27, 1971). Experiments havedemonstrated that this compound effectively inhibits the polymerizationof microtubles of mammalian cells without undue toxicity and, as such,is an effective anti-tumorigenic agent. Clinical trails identified taxolas extremely effective against refractory ovarian, breast and othercancers. As such, taxol is a breakthrough in chemotherapy because of itsrather unique, but basic mechanism of action being fundamental distinctfrom conventional chemotherapeutic agents (L. A. Rowinsky et al., J.Natl. Cancer Instit. 82:1247-59, 1990).

The most daunting variable in the taxol equation so far is supply. Ittakes three to six, 100 year old Pacific yews to treat one patientbecause average yields of taxol are low (Witherup et al., 1990). Theproduction of an amount of taxol needed for treatment and testing willrequire the destruction of tens of thousands of yews. The yew populationhas been rendered nearly extinct by logging and as the number of Pacificyews dwindles, medical research must look for other forms of supply fortaxol. The usefulness of taxol, as well as many other compounds whichmay be propagated or harvested in plant cells, has fueled an interest inculturing taxus and other plant cells.

The culturing of plant cells for their biosynthetic ability posesspecial problems for current technology. Prolonged culturing of plantcells often results in a loss of biosynthetic ability which had beenpresent in the original isolates (Dhoot et al., Ann. Bot. 41:943-49,1977; Barz et al., Ber. Dtsch. Bot. Ges. 94:1-26, 1981). Phenotypicalterations also arise which further complicate cell culturing. Aprotocol for freezing plant cells, especially taxus cells, is animportant step in the development of biosynthetic methods for productionof useful plant alkaloids such as taxol.

SUMMARY OF THE INVENTION

The present invention overcomes the problems and disadvantagesassociated with current strategies and designs and provides novelmethods for cryopreservation and for the recovery of viablecryopreserved plant cells.

One embodiment of the invention is directed to methods for thecryopreservation of plant cells. Plant cells, which may be gymnospermsor angiosperms, are pretreated with a cryoprotective agent and astabilizer, and acclimated to a reduced temperature. Acclimated cellsare loaded with a loading agent which may be the same as the vitrifyingagent and the loaded cells vitrified with a vitrification solution.Vitrified plant cells are frozen at cryopreservation temperatures, suchas, between about -70° C. to about -200° C. or less.

Another embodiment of the invention is directed to methods forcryopreserving plant cells. Plant cells to be cryopreserved arepretreated with a cryoprotective agent and a stabilizer, and acclimatedto a reduced temperature. Acclimated plant cells are vitrified andfrozen at a cryopreservation temperature.

Another embodiment of the invention is directed to methods forcryopreserving plant cells. Plant cells to be cryopreserved are culturedin media comprising a vitrifying agent and a stabilizer at a reducedtemperature for a first period of time. The cultured plant cells arefurther cultured in media containing an increased concentration of thevitrifying agent for a second period of time. Plant cells vitrified inthe higher concentration of vitrifying agent are frozen at acryopreservation temperature.

Another embodiment of the invention is directed to methods forcryopreserving plant cells. Plant cells to be cryopreserved arelyophilized by vacuum evaporation and vitrified in a vitrifyingsolution. Lyophilization removes about 60% of the water from the cellsand in combination with vitrification can remove up to about 95%. Thevitrified and lyophilized plant cells are frozen and stored at acryopreservation temperature by, for example, quenching the cells intoliquid nitrogen.

Another embodiment of the invention is directed to methods forrecovering plant cells from cryopreservation. Plant cells arecryopreserved according to the methods of the invention. Thawed plantcells are warmed to a temperature above freezing and incubated in amedia comprising a cryoprotective agent and a stabilizer. The osmoticagent is removed and viable plant cells recovered.

Another embodiment of the invention is directed to methods forrecovering cryopreserved plant cells from cryopreservation.Cryopreserved plant cells are thawed to a temperature above freezing andincubated in media comprising an cryoprotective agent and a stabilizer.The cryoprotective agent is removed such as by dilution of the mixtureor pelleting of the cells and viable plant cells recovered.

Another embodiment of the invention is directed to viable plant cellswhich have been cryopreserved by the method of the invention.Cryopreserved plant cells are not significantly genetically orphenotypically altered by cryopreservation.

Another embodiment of the invention is directed to methods forrecovering cryopreserved plant cells in suspension. Cryopreserved plantcells are thawed to a temperature above freezing. Thawed plant cells areincubated in liquid suspension and viable cells recovered in liquidmedia without a need for solid or semi-solid culture.

Another embodiment of the invention is directed to viable plants andplant cells cryopreserved and to viable plants and plant cells recoveredby the methods of the invention. Cells are not significantlygenotypically or phenotypically altered by the cryopreservation processand have a high proportion of survival.

Other embodiments and advantages of the invention are set forth, inpart, in the description which follows and, also in part, will beobvious from this description or may be learned from the practice of theinvention.

DESCRIPTION OF THE FIGURES

FIG. 1 Schematic of various cryopreservation and recovery protocols.

FIG. 2 Procedure for cryopreservation of Taxus cells.

FIG. 3 Biomass increase in a Taxus chinensis suspension culture lineK-1.

FIG. 4 Chromatograms of (A) cells cryopreserved for 6 months incomparison with (B) non-cryopreserved cells.

FIG. 5 Chromatograms of (A) cells cryopreserved for 6 months incomparison with (B) non-cryopreserved cells.

FIG. 6 Analysis of genetic stability of cryopreserved cells by Southernblot.

FIG. 7 Analysis of genetic stability of cryopreserved by PCR.

DESCRIPTION OF THE INVENTION

As embodied and broadly described herein, the present invention isdirected to methods for the cryopreservation of plant cells, methods forthe recovery of cryopreserved plant cells and viable plant cells whichhave been successfully recovered from cryopreservation.

Plant cells are increasingly useful for the production of recombinantprotein or unique products and chemical agents which are specific toplants or to the enzymatic pathways of plant cells. Plant cells such ascallus cultures can be maintained in a continuous state through repeatedsub-culturing. However, sub-culturing frequently results in increasedploidy, an increased risk of contamination, an accumulation ofspontaneous mutations, a decline and loss of morphogenetic potential, areduction of biosynthetic capacity for product formation, a reversion ofselected lines to wild-types, aging and transformation infinite celllines and unintentional selection of undesirable phenotypes. Each ofthese factors can severely impede the exploitation of cell culturesystems for commercial production of valuable compounds.

While animal tissue cultures cells have been routinely cryopreserved formany years, similar cryopreservation techniques for plant cells hasproven to be far more difficult. Plant cells and, in particular, plantcells in culture, exhibit an array of heterogeneity with respect togrowth rate, doubling time, mitotic index, cell synchrony, nuclear tocytoplasmic ratio and extent of vacuolation. Cells present in any giveculture also exhibit a variety of physiological and morphologicalvariations. Further, plant cell suspensions and adherent cell culturesrequire different protocols for cryopreservation. In addition, ifperformed improperly, cryopreservation can induce the very mutationswhich the process is attempting to prevent.

Surprisingly, it has been discovered that by using a series of steps andspecific agents, plant cells of most any genus and species can becryopreserved and successfully recovered. These methods are based on theobservations that successful plant cell cryopreservation involves theremoval of substantial amounts of water from the cells and that underappropriate conditions, significant amounts of water can be removedwithout seriously effecting cell viability. Cryopreservation protocolsdeveloped are highly successful at storing, maintaining and retrievingviable cells in a routine and reproducible manner. These protocols canbe established in routine unit operations to create germplasm storageand cell bank management systems. In addition, cells can be recoveredentirely in liquid suspension, a process previously thought not to bepossible with plant cultures. Products harvested from cryopreservedcells do not significantly vary from the original or parent cell asthere has been little if any phenotypic or genotypic drift, particularlywith respect to respect to growth and viability, product formation andcell biomass proliferation.

One embodiment of the invention is directed to methods for thecryopreservation of plant cells. These methods are surprisinglyreproducible and applicable to many types of plant cells. As such, theywill be markedly useful for the production of materials which requiregovernment or agency standards of reproducibility.

The basic process involves the removal of substantial amounts of waterfrom the cell by a combination of pretreating, cold acclimating,loading, vitrifying, lyophilizing and freezing steps. A wide variety ofcombinations of these steps is possible and every step is notnecessarily required for the successful cryopreservation and recovery ofviable plant cells. Some of the possible combinations of steps areschematically depicted in FIG. 1, although it is understood that thesevariations are only exemplary.

Most any plant cell can be successfully cryopreserved and recoveredusing these processes including the gymnosperms and the angiosperms.Specific types of gymnosperms which can be cryopreserved include speciesof the genera Abies (firs), Cypressus (cypresses), Ginkgo (maidenhairtree), Juniperus (juniper), Picea (spruce), Pinus (pine), Pseudotsuga(Douglas fir), Sequoia, Taxus (yew), Tsuga (hemlock) or Zamia (cycad).Some of the more useful species of Taxus include T. baccata, T.brevifolia, T. canadensis, T. chinensis, T. cuspidata, T. floridana, T.globosa, T. media, T. nucifera and T. wallichiana. Angiosperms which canbe preserved include monocotyledon plant cells and dicotyledon plantcells. Monocotyledon plant cells include a variety of species of thegenus Avena (oat), Cocos (coconut), Dioscorea (yam), Hordeum (bareley),Musa (banana), Oryza (rice), Saccharum (sugar cane), Sorghum (sorghum),Triticum (wheat) and Zea (corn). Dicotyledon plants include species ofthe genus Achyrocline, Atropa, Brassica (mustard), Berberis, Capsicum,Catharanthus, Conospermum, Datura, Daucus (carrot), Digitalis,Echinacea, Eschscholtzia, Glycine (soybean), Gossypium (cotton),Hyoscyamus, Lycopersicum (tomato), Malus (apple), Medicago (alfalfa),Nicotiana, Panax, Pisum (pea), Rauvolfia, Ruta, Solanum (potato) andTrichosanthes.

Plant cells may be freshly harvested specimens from the field as newgrowth needles, leaves, roots, bark, stems, rhizomes, callus cells,protoplasts, cell suspensions, organs or organ systems, meristems suchas apical meristems, seeds or embryos. Generally, low passage cells andprimary cultures show greater ultimate viability in culture or uponrecovery from cryopreservation. Alternatively, sample cells may beobtained from established in vitro cultures. Cultures may have been longestablished or only recently adapted to in vitro conditions of, forexample, specific temperatures, particular light intensities or specialgrowth or maintenance mediums. Such cells may be maintained assuspension cells or by growth on semi-solid nutrient medium.

Suspension cultures can be derived from callus cultures of a Taxusspecies or from thawed cryopreserved cells of a Taxus species. Lowpassage primary cell lines are very valuable to preserve as thesecultures may exhibit unique characteristics which are lost with extendedtime in culture. Many of these cell lines express diterpenoids such asthe diterpenoid alkaloid taxane, the ester side chain modified taxane,taxol (molecular weight 853), and a variety of other modifications oftaxane (baccatin or 10-deactylbaccatin).

Taxus cells for culture can be obtained from any plant material.Collections can be made from over North America as well as othercontinents. Tissue from any part of the plant including bark, cambium,needles, stems, seeds, cones and roots, can be selected and adapted forcell culture. Needles and meristematic regions of plants, especially oneto three month old new growth needles are preferred for initiating cellcultures. For example, selected plant tissue is surface-sterilized byimmersion in a four liter 10% solution of bleach with 10 drops of Tween20 added for 20 minutes. Tissues explants are cut to very small sizesand cultured.

Taxus cultures typically exhibit variability in growth morphology,productivity, product profiles and other characteristics. As individualcell lines vary in their preferences for growth medium constituents,many different growth media may be used for induction and proliferationof the cell culture. Methods of sterilization, initiation of callusgrowth, and suspension growth, as well as suitable nutrient media, arewell-known to those of ordinary skill in the art.

Taxus suspension cultures are capable of rapid growth rates and highcell densities. Initial cultures of various Taxus species aresub-cultured by transfer into suitable media containing, for example,macro and micro nutrients, organic salts and growth hormones. Liquidcultures are exposed to air and preferably agitated to aerate the mediumor air may be bubbled in the medium. Cultures are maintained at atemperature of about 20° C. and at a pH of between about 3 to about 7,and preferably of between about 4 to about 6. Such cultures can beharvested by removal of the growth medium, for example, by filtration orcentrifugation. Cells with the highest viability are those at the earlylag or early cell division growth phases or recently passed through celldivision or mitosis. Generally, 4-10 day old cell suspension of a Taxusspecies in growth medium, preferably 5-8 day old cell suspensions ingrowth medium, and more preferably 6-7 day old cell suspensions of aTaxus species in growth medium, are suitable for use.

Each of the basic steps of cryopreservation are presented below:

Pretreatment

Plant cells to be cryopreserved can be pretreated with agents thatincrease cellular viability by removing harmful substances secreted bythe cells during growth or cell death from the culture medium. Theseagents, referred to as stabilizers herein, remove both naturallyoccurring and artificially introduced substances from the culturemedium. Stabilizers are anti-oxidants or radical scavenger chemicalsthat neutralize the very deleterious effects attributable to thepresence of active oxygen species and other free radicals. Suchsubstances are capable of damaging cellular membranes, both internal andexternal membranes, such that cryopreservation and recovery of areseriously compromised. If these substances are not removed or renderedotherwise rendered ineffective, their effects on viability arecumulative over time severely limiting practical storage life.Furthermore, as cells die or become stressed, additional harmfulsubstances are released increasing the damage and death of neighboringcells.

Useful stabilizers include those chemicals and chemical compounds whichsequester highly reactive and damaging molecules such as oxygenradicals. Specific examples of these radical scavengers andanti-oxidants include reduced glutathione, 1,1,3,3-tetramethylurea,1,1,3,3-tetramethyl-2-thiourea, sodium thiosulfate, silver thiosulfate,betaine, N,N-dimethylformamide, N-(2-mercaptopropionyl) glycine,β-mercaptoethylamine, selenomethionine, thiourea, propylgallate,dimercaptopropanol, ascorbic acid, cysteine, sodium diethyldithiocarbomate, spermine, spermidine, ferulic acid, sesamol,resorcinol, propylgallate, MDL-71,897, cadaverine, putrescine, 1,3- and1,2-diaminopropane, deoxyglucose, uric acid, salicylic acid, 3- and4-amino-1,2,4-triazol, benzoic acid, hydroxylamine and combinations andderivatives of such agents.

Stabilizers are preferably incubated with plant cells prior to freezing,although their presence during recovery, thawing and regrowth, may alsobe desirable. Incubations can be performed for hours or days as theagents themselves are generally not harmful to the cells and may evenincrease viability. Some of the more sensitive plant cell lines mayrequire longer treatments while others shorter. The exact period ofincubation can be easily determined empirically. Preferably, plant cellsare cultured in growth medium with the stabilizer or a combination ofstabilizers for about 1 to about 10 days, more preferably for about 1 toabout 7 days and even more preferably about 3 days. This is typicallysufficient time for most of the most damaging substances in the mediumto be eliminated or at least reduced to levels which are no longerharmful to the cells.

Incubations can be performed in liquid or semi-solid mediums such asgrowth medium, medium that encourages metabolism and cell proliferation,or maintenance medium, medium that provides a balance of salts andnutrients without necessarily encouraging cell growth. As the cells arebeing prepared for cryopreservation, it is sometimes desirable toincubate in maintenance medium to reduce the metabolic processes of thecells.

Pretreatment can be performed at room temperature or at temperatureswhich the plant cells are typically cultured. Preferably, pretreatmentis performed at about room temperature (20° C.) for ease of handling andas most plant cells are fairly stable at room temperature. Stabilizerscan be added directly to the medium and replenished as necessary duringthe pretreatment process. Stabilizer concentrations are particular tospecific stabilizers, but are generally used at between about 1 μM toabout 1 mM, although more or less of specific stabilizing agents wouldnot be uncommon.

Pretreatments may also involve incubating cells in the presence of oneor more osmotic agents. Examples of useful osmotic agents include sugarssuch as saccharides and saccharide derivatives, ammo or imino acids suchas proline and proline derivatives, or combinations of these agents.Some of the more useful sugars and sugar derivatives are fructose,glucose, maltose, mannitol, sorbitol, sucrose and trehalose. Osmoticagents are utilized at a concentration that prepares cells forsubsequent loading, lyophilization and/or vitrification. Concentrationscan vary greatly between different agents, but are generally betweenabout 50 mM to about 2 M. Preferably, osmotic agents concentration inmedia are between about 0.1 M to about 0.8 M, and more preferably atbetween about 0.2 M to about 0.6 M. Alternatively, the osmotic agent isemployed as an aqueous solution at a concentration of between about 1%to about 10%, by weight.

Cold Acclimation

During or at some time after pretreatment, cells to be cryopreserved maybe acclimated to a temperature which is reduced from culturingtemperatures, but above freezing. This prepares cells for thecryopreservation process by significantly retarding cellular metabolismand reduces the shock of rapid temperature transitions through some ofthe more critical temperature changes.

Acclimation results in the accumulation of endogenous solutes thatdecreases the extent of cell dehydration at any given osmatic potential,and contributes to the stabilization of proteins and membranes duringextreme dehydration. In addition, cold adaptation interactssynergistically with the vitrifying agents and results in alterations inthe liquid conformation of the cellular membranes, that increasetolerance to both osmotic exclusions and dehydration.

Preferably, acclimation temperatures are between about 1° C. to about15° C., more preferably between about 2° C. to about 10° C. and evenmore preferably about 4° C. Cells may be gradually, in a step-wise orcontinuous manner, or rapidly acclimated to the reduced temperature.Techniques for acclimation are well known to those of ordinary skill andinclude commercially available acclimators. Gradual acclimationcomprises reducing incubation temperatures about 1° C. per hour untilthe target temperature is achieved. Gradual acclimation is most usefulfor those cells considered to be most sensitive and difficult tocryopreserve.

Suspension cells can be in the late lag or early cell division phases toachieve the greatest survival rates on freezing and thawing. Cellsbeyond these phases exhibit higher degrees of vacuolation anddifferentiation, and are larger in size, thus enhancing the risk offreezing injury and decreasing survival rates on freezing and thawing.

Loading

Loading involves the equilibration of cells in a solution of one or morecryoprotectants. Agents utilized during loading may be referred to asloading agents. Useful loading agents may include one or moredehydrating agents, permeating and nonpermeating agents, and osmoticagents. Suitable agents for loading include agents which induce celldehydration upon introduction to a cell suspension. Both permeatingagents such as DMSO and ethylene glycol, and a combination of permeatingand nonpermeating osmotic agents such as fructose, sucrose or glucose,and sorbitol, mannitol or glycerol can be used. This step increasessolute concentration of the cytosol by introducing moderateconcentrations of cryoprotective agents, generally at between about 0.5M to about 2 M or between about 5% to about 20%, by weight, into thecytosol. Preferably, the loading agent is employed as an aqueoussolution at between about 0.05 M to about 0.8 M or from about 1% toabout 10% by weight. Loading may comprise incubating the plant cells ina solution comprising between about 0.5% to about 10% by weight of avitrifying agent. To minimize the time required for equilibration,loading is usually performed at about room temperature, although optimaltemperature and other conditions for loading will preferably matchconditions such as medium, light intensities and oxygen levels thatmaintain a viable cell culture.

In the loading step, single cryoprotective agents or combinations ofdifferent cryoprotective agents can be added directly to the incubationmedium. Cells are incubated in this solution for a period of time toequilibrate intracellular and extracellular concentrations of the agent.In addition, loading also removes a small, but significant amount ofwater from the cell. This prepares the cell for subsequent vitrificationand/or lyophilization by minimizing the shock of sudden intracellularwater loss.

After loading, growth medium containing the cryoprotective agent can beremoved or, if the following agent (vitrifying agent) to be utilized isthe same or a similar agent, the loading agent can remain and theconcentration simply increased for vitrification. The loading agent andvitrifying agent may be the same and, further, loading and vitrifyingmay be performed substantially simultaneously.

Vitrification

Cells to be cryopreserved are vitrified following pretreatment, loadingand/or lyophilization. There are several advantages of the vitrificationprocedures. By precluding ice formation in the system, the need tooptimize the complex set of variables which lead to ice formation iseliminated. Further, specimens can be plunged directly in liquidnitrogen, the procedure does not require extensive equipment requiredfor controlled cooling. The vitrification procedure also offers thegreatest potential for developing cryopreservation procedures forcomplex tissues and organs that are comprised of several different typesof cells.

Vitrification procedures involve osmotic dehydration of the cells orspecimens prior to quenching in liquid nitrogen. In vitrificationprocedures, cell dehydration is effected by direct exposure toconcentrated solutions prior to cooling in liquid nitrogen. Under idealconditions the cells or organs can be cooled at extremely rapid rateswithout undergoing intracellular ice formation. As a result, all of thefactors that affect intracellular ice formation are obviated and thereare several practical advantages of the vitrification procedures incomparison to conventional cryopreservation procedures. Vitrificationoffers the greater potential for developing cryopreservation proceduresfor complex tissues and organs. By precluding significant ice formationin the system, the vitrification procedure is operationally more complexthan conventional cryopreservation procedures. Further, vitrificationallows for the use of ultra-rapid cooling rates to circumvent problemsof chilling sensitivity of some specimens. No specialized or expensiveequipment is required for controlled cooling rates.

Vitrification is a cryogenic method wherein a highly-concentratedcytosol is super-cooled to form a solid, metastable glass withoutsubstantial crystalization. The major difficulty in cryopreservation ofany cell is the formation of intracellular ice crystals during bothfreezing and thawing. Excessive ice crystal formation will lead to celldeath due to disruption of cellular membranes and organelles. One methodto prevent ice crystal formation is to freeze the cells rapidly suchthat the ice crystals formed are not large enough to cause significantdamage. When a cell with a low water content is frozen rapidly, itvitrifies. Vitrification by rapid freezing is not possible with cellssuch as plant cells which containing a high water content. To vitrifyhigh water content cells, freezing point reduction agents and icecrystal inhibitors is needed in addition to rapid freezing forvitrification. A properly vitrified cell form a transparent frozenamorphous solid consisting of ice crystals too small to diffract light.If a vitrified cell is allowed to warm to about -40° C., it may undergodevitrification. In devitrification, ice crystals enlarge andconsolidate in a process which is generally detrimental to cellsurvival. Vitrification solutions enhance vitrification of cells uponfreezing or retard devitrification upon thawing.

Most cryopreservation solutions can transform the subject material intoa glass or glass-like material provided cooling rates are sufficientlyrapid to prevent the nucleation and growth of ice crystals, with thecritical cooling rate dependent on the solute concentration. Similarly,vitrification of the cells can be effected if the cytosol issufficiently concentrated. In cryopreservation procedures, this isachieved by dehydrating the cells in extremely concentrated solutionsprior to quenching in liquid nitrogen. With cryopreservation, thecytosol is concentrated to the level required for vitrification byplacing the specimens in a concentrated solution, such as about 4 M toabout 10 M, or at between about 25% to about 60%, by weight,concentration of a cryoprotective, the vitrifying agent. This producesan extreme dehydration of the sample cells. Solutions in excess of 7 Mwill typically remove more than 90% of the osmotically active water fromthe cells, however, precise concentrations for each agent can beempirically determined. Vitrifying agents which may be used includeDMSO, propylene glycol, mannitol, polyethylene glycol, ethylene glycol,butanediol, formamide, propanediol and mixtures of these materials.

Suitable vitrification solutions include culture medium with DMSO(1-50%), propylene glycol (about 5-25%), glycerol (about 0-50%),PEG-8000 (about 5-20%), ethylene glycol (about 10-75%), ethyleneglycol/sorbitol (about 60/20 weight percent to about 10/60 weightpercent), and ethylene glycol/sorbitol (about 40/30 weight percent).Ethylene glycol/sorbitol is preferred and can be employed atconcentrations of, for example, 50/30%, 45/35%, 40/40%, 40/30%, 30/50and, preferably, 30/40%. Such vitrification solutions can be utilized attemperatures from about 1° C. to about 8° C., preferably at atemperature of from 2° C. to 6° C., and more preferably at about 4° C.

To minimize the injurious consequences of exposure to vitrificationsolutions, dehydration may be performed at about 0° C. to about 40° C.,with the time of exposure as brief as possible. Under these conditions,there is no appreciable influx of additional cryopreservation into thespecimens because of the difference in the permeability coefficient forwater and solutes. As a result, the specimens remain contracted and theincrease in the cytolic concentration required for vitrification isattained by dehydration. However, equilibrium (loading) of the cellswith cryoprotectants is not always required for successful vitrificationof plant cells or organs. For example, in some cases, preculturing withloading agents achieve the same purposes as the loading step.

In those instances in which loading is required, it primarily serves toprevent dehydration-induced destabilization of cellular membranes andpossibly proteins. However, in the absence of a loading step, there canbe less survival of cells following the dehydration and cooling/warmingsteps. Thus, intracellular ethylene glycol or other cryoprotectantsduring the loading step not only favors vitrification of the cellsduring cooling, but also protects cells against injury during thedehydration step.

Lyophilization

Lyophilization is directed to reducing the water content of the cellsbefore cryopreservation by vacuum evaporation. Vacuum evaporationinvolves placing the cells in an environment with reduced air pressure.Depending on the rate of water removal desired, the reduced ambientpressure operating at temperatures of between about -30° C. to -50° C.may be at 100 torr, 1 torr, 0.01 torr or less. Under conditions ofreduced pressure, the rate of water evaporation is increased such thatup to 65% of the water in a cell can be removed overnight. With optimalconditions, water removal can be accomplished in a few hours. The heatloss during evaporation maintains the cells in a chilled state. Bycareful adjustment of the vacuum level, the cells may be maintained at acold acclimation temperature during the vacuum evaporation process. Astrong vacuum, while allowing rapid water removal exposes the cells tothe danger of freezing. Freezing may be controlled by applying heat tothe cells directly or by adjustment of the vacuum level. When the cellsare initially placed in the evaporative chamber, a high vacuum may beapplied because the residue heat in the cells will prevent freezing. Asdehydration proceeds and the cell temperature drops, the vacuum may bedecreased or heating may be applied to prevent freezing. The semi-drycells may have a tendency to scatter in an evaporative chamber. Thistendency is especially high at the end of the treatment when anairstream is allowed back into the chamber. If the air stream proximatesthe semi-dry cells, it may cause the cells to become airborne and causecross contamination of the samples. To prevent such disruptions,evaporative cooling may be performed in a vacuum centrifuge wherein thecells are confined to a tube by centrifugal force while drying. Theamount of water removed in the process may be monitored periodically bytaking dry weight measurement of the cells. When the desired amount ofwater is removed, vitrification solution may be added directly to thesemi-dry cells for a period to time prior to direct freezing in liquidnitrogen.

Freezing

Plant cells, which may have been pretreated, loaded, vitrified and/orlyophilized, are preserved by freezing to cryopreservation temperatures.The freezing step should be sufficiently rapid to prevent the formationof large ice crystals which are detrimental to the cell's survival.Cells may be directly frozen, that is brought directly into contact withan agent already at cryopreservation temperature. Direct methods includedripping, spraying, injecting or pouring cells directly into a cryogenictemperature fluid such as liquid nitrogen or liquid helium. Cells mayalso be directly contacted to a chilled solid, such as a liquid nitrogenfrozen steel block. The cryogenic fluid may also be poured directly ontoa container of cells. The direct method also encompasses contact cellswith gases, including air, at a cryogenic temperature. A cryogenic gasstream of nitrogen or helium, may be blown directly over or bubbled intoa cell suspension. Indirect method involved placing the cells in acontainer and contacting the container with a solid, liquid, or gas atcryogenic temperature. Proper containers include plastic vials, glassvials, ampules which are designed to withstand cryogenic temperatures.The container for the indirect freezing method do not have to beimpermeable to air or liquid. For example, a plastic bag or aluminumfoil are adequate. Furthermore, the container may not necessarily beable to withstand cryogenic temperatures. A plastic vial which cracksbut remain substantially intact under cryogenic temperatures may also beused. Cells may also be frozen by placing a sample of cells on one sideof a metal foil while contacting the other side of the foil with a gas,solid, or liquid at cryogenic temperature. Freezing should besufficiently rapid to inhibit ice crystal formation. The freezing timeshould be around 5 minutes or 4 minutes, 3 minutes, 2 minutes, or oneminute or less. The critical freezing time should be measured from theframe of reference of a single cell. For example, it may take 10 minutesto pour a large sample of cells into liquid nitrogen, however theindividual cell is frozen rapidly by this method.

Thawing

Another embodiment of the invention is directed to methods for thawingcryopreserved cells. Proper thawing and recovery is essential to cellsurvival. As the temperature of the cryopreserved cells is increasedduring thawing, the small ice crystals will consolidate and increase insize in a process termed devitrification. Large intracellular icecrystals are generally detrimental to cell survival. To preventdevitrification, cryopreserved cells should be thawed as rapidly aspossible. The rate of heating may be at least about 30° C. per minute to60° C. per minute. More rapid heating rates of 90° C. per minute, 140°C. per minute to 200° C. or more per minute can also be used. Whilerapid heating is desired, plant cells have reduced ability to surviveincubation temperature significantly above room temperature. To preventoverheating, the cell temperature should be carefully monitored. Anyheating method can be employed including conduction, convection,radiation, electromagnetic radiations or combinations. Conductionmethods involves immersion in water baths, placement in heat blocks ordirect placement in open flame. Convection methods involve the use of aheat gun or an oven. Radiation methods involve, for example, heat lampsor ovens such as convection or radiation ovens. Electromagneticradiation involves the use of microwave ovens and similar devices. Somedevices may heat by a combination of methods. For example, an oven heatsby convection and by radiation. Heating should be terminated as soon asthe cells and the surrounding solutions are in liquid form, which shouldbe above 0° C. Cryopreserved cells are often frozen in the presence ofagents which depresses the freezing point. When these agents arepresent, the frozen cells may liquify at a temperature below 0° C. suchas -10° C., -20° C., -30° C. or -40° C. Thawing of the cryopreservedcells may be terminated at any of these temperature or at a temperatureabove 0° C.

Post-Thawing

Dilution of the vitrification solution and removal of cryopreservativefrom the cells, referred to as unloading, should be performed as rapidlyas possible and as quickly as possible subsequent to thawing of thecryopreserved cell sample. Due to the high intracellular concentrationsof cryopreservative, it is preferred to effect the dilution of thesuspending medium while minimizing osmotic expansion. Therefore,dilution of the suspending medium and afflux of the cryopreservationfrom within the sample specimen is usually accomplished by dilution inan hypertonic medium or a step-wise dilution.

Thawed cells can be gradually acclimated to growth conditions tomaximize survival. Vitrification agents may be cytotoxic, cytostatic, ormutagenic and should be removed from the thawed cells at a rate whichwould not harm the cells. A number of removal methods may be used suchas resuspension and centrifugation, dialysis, serial washing,bioremediation and neutralization with chemicals and electromagneticradiation. The rapid removal of some vitrification solutions and osmoticagents may increase cell stress and death and thus the removal step mayhave to be gradual. The removal rate may be controlled by serial washingwith solutions which contain less osmotic or vitrification agents. Othermethod to reduce the removal rate include dialysis with less permeablemembranes, serial growth on semisolid or liquid media containing lessand less vitrification agents. Other methods include gradual dilutions,dialysis, bioremediation, neutralization and catalytic breakdown of thecryogenic agent.

Thawing and post-thaw treatments may be performed in the presence ofstabilizers to ensure survival and minimize genetic and cellular damage.The stabilizer may reduce, eliminate or neutralize damaging agents whichresults from cryopreservation. Such damaging agents include freeradicals and oxidizers. Survival and regrowth rates are surprisinglyenhanced with the addition of stabilizers during the thawing and postthawing steps.

Cells can be regrown in suitable media after levels of osmotic orvitrification agents are reduced to an acceptable level. One method forregrowth involves placing the thawed cells in semisolid growth media,such as agar plates, until a callus is formed. Cells may be recoveredfrom the callus and grown in liquid culture. Alternatively, callus cellsmay be induced to grow shoots and roots by placement in semisolid mediacontaining the appropriate hormones. Callus cells with shoots and rootsmay be gradually acclimated to grow on sterile soil in a greenhouseuntil a plant develops. The greenhouse plant may be acclimated to grownoutside a greenhouse in its natural environment. Alternatively, a cellmay be thawed and regrown without the use of semi-solid media. That is,after removal of osmotic and vitrification agents, the cells may beplaced directly into liquid media for regrowth.

One method to implement cryopreservation of plant cells, for example,taxol producing taxus cells, is schematically represented in FIG. 2.Briefly, a callus cell growth from a primary isolate or from a cellculture is adapted for culture in liquid growth medium. After the cellshave adjusted to liquid culture, they are transferred to a pretreatmentgrowth medium containing an osmotic agent and a stabilizer for 24 to 72hours. The loaded cells are subsequently cold acclimated by theincubation of the pretreatment culture at 4° C. for 1 to 4 hours. Aftercold acclimation, the cells are transferred to a centrifuge vial andsubjected to mild centrifugation which pellets the cells without damage.The supernatant, comprising the pretreatment media with osmotic agentsand the stabilizer is aspirated from the cell pellet and discarded. Aprechilled solution, comprising a stabilizer and a vitrification agent,is added to the cell pellet and the cells are gently resuspended. After3 minutes of treatment with the vitrification solution, the cells areplaced into archival cryogenic storage by immersion of the vialcontaining the cells in liquid nitrogen. Cryogenically preserved cellsare stored in liquid nitrogen for a period from about 30 minutes to manyyears. To revive the cells, the vial containing the cryopreserved cellsis rapidly transferred from liquid nitrogen to a 40° C. water bath. Thevial is removed from the 40° C. bath when the contents are liquified. Analiquot of the thawed cells is inspected for immediate viability by dyeexclusion analysis. The remaining cells are washed by a series of coldgrowth medium containing a stabilizer and a progressively lowerconcentrations of osmotic agents. After sufficient osmotic andvitrification agents are removed from the cells by serial washes, thecells are transferred directly into liquid culture. Alternatively, cellsare placed on a plating paper and transferred to a series of semi-solidmedium with a stabilizer and a decreasing amount of osmotic andvitrification agents. This serial plating is continued until the cellshave adjusted to growth on semi-solid medium in the absence of osmoticand vitrification solutions.

Another embodiment of the invention is directed to plant cells whichhave been cryopreserved by the methods described above. Cells may be ofany genus or species disclosed or which the cryopreservation methods canbe applied. Cryopreserved cells may be maintained at temperaturesappropriate for cryo-storage. Preferably, cells are maintained in liquidnitrogen (about -196° C.), liquid argon, liquid helium or liquidhydrogen. These temperatures will be most appropriate for long termstorage of cells, and further, temperature variations can be minimized.Long term storage may be for months and preferably for many yearswithout significant loss of cell viability upon recovery. As theinvention also relates to efficient methods for recovery ofcryopreserved cells, relatively large portions of cell samples may belost without loss of the entire sample. Cells or plants can bepropagated from those cells that remain. Short term storage, storage forless than a few months, may also be desired wherein storage temperaturesof -150° C., -100° C. or even -50° C. may be used. Dry ice (carbondioxide) and commercial freezers may be used to maintain suchtemperatures.

Another embodiment of the invention is directed to plants and plantcells which have been revived by the cryopreservation recovery methodsdescribed above. These cells may also be of any of the genus or speciesdisclosed herein or a genus or species to which the methods ofcryo-recovery have been applied. Cells may be the original cells whichwere cryopreserved or cells which have proliferated from such cells.

Another embodiment of the invention is directed to methods and kits forthe transportation and thawing of cryopreserved cells. Cellscryopreserved by this method may be stored in a central repository forsubsequent retrieval. For increased safety against accidental loss, eachcell line frozen may be stored in a number of locations. Duringretrieval, a cryovial containing the cryopreserved cells may be shippedin a suitable container to the recipient. Suitable container are thosewhich can maintain cryopreservation temperature during shipment. Allcells can be shipped at temperatures sufficiently low for long termstorage with portable cryopreservation agents such as liquid nitrogen.Cells destined for immediately thawing may be shipped in dry ice toreduce cost. A kit for the retrieval of cells from a repository mayinclude a vial of cryopreserved cells, sufficient media with theappropriate concentrations of osmotic agents, vitrification solutions,and stabilizers for serial washes. Alternatively, in place or inaddition to the wash solution, the cells may be shipped with a pluralityof semisolid growth media comprising a stabilizer and decreasing amountsof osmotic and vitrification solutions. After thawing, the cells areeither washed and used immediately or they may be placed on thesemisolid media to gradually remove the vitrification and osmoticagents. The transport kit may further include reagents for an post thawviability assay and a reference DNA sample for comparison with DNA fromthe thawed cells to determine genetic stability.

The following experiments are offered to illustrate embodiments of theinvention and should not be viewed as limiting the scope of theinvention.

EXAMPLES Example 1 Callus Initiation and Proliferation

Taxus needles were collected from wild and cultivated plants. Plantmaterial was washed in a diluted soap solution, rinsed extensively withdistilled water and surface sterilized in a chlorous solution (1%hypochlorite, pH 7) for 10 minutes. Under sterile conditions, thematerial was rinsed 3 times with sterile distilled water. Needles werecut in a 1% polyvinylpyrrolidone (PVP) solution with 100 mg/L ascorbicacid. Needles were placed with the cut end in semisolid medium E andincubated at 24° C.±1° C. in the dark. Cultures were monitored daily andthose with signs of contaminating microorganisms were discarded.Substantial callus formation was observed and the callus was separatedfrom the explant by 20 days and placed on the various callusproliferation media listed in Table 1. Calli of Taxus chinensis weretransferred to medium D (Table 1). This procedure resulted in callusinduction in over 90% of the explants. The same procedure wassuccessfully used to initiate cultures of T. brevifolia, T. canadensis,T. cuspidata, T. baccata, T. globosa, T. floridana, T. wallichiana, T.media and T. chinensis. Calli removed from the explant were cultivatedat 24° C. in the dark. Healthy parts of the callus were transferred tofresh medium every 10 days. The preferred growth and maintenance mediafor the invention are listed:

                                      TABLE 1                                     __________________________________________________________________________    Chemical Composition of Various Growth Medium                                 CHEMICAL                                                                      INGREDIENT A    B    C    D    E    F    G    H                               __________________________________________________________________________    Ammonium Nitrate                                                                         --   --   --   --   --   400  500  400                             Ammonium Sulfate                                                                         134  --   33.5 134  67   --   134                                  Boric Acid 3    1.5  0.75 3    1.5  0.75 6.2  1.5                             Calcium Chloride                                                                         113.2                                                                              --   28.31                                                                              113.24                                                                             56.62                                                                              72.5 113.24                                                                             72.5                            (anhydrous)                                                                   Calcium Chloride 2H.sub.2 O                                                              --   0    50   --   --   --   --   --                              Calcium Nitrate 4H.sub.2 O                                                               --   208.4                                                                              --   --   --   386  --   386                             Cobalt Chloride 6H.sub.2 O                                                               0.03 --   0.006                                                                              0.025                                                                              0.0125                                                                             --   0.025                                                                              --                              Cupric Sulfate 5H.sub.2 O                                                                0.03 0.01 0.006                                                                              0.025                                                                              0.0125                                                                             0.25 0.025                                                                              0.25                            Na.sub.2 EDTA 2H.sub.2 O                                                                 37.3 --   9.32 37.3 18.65                                                                              37.3 37.3 37.3                            Ferric Sulfate                                                                           --   2.5  --   --   --   --   --   --                              Ferrous Sulfate 7H.sub.2 O                                                               27.8 --   6.95 27.8 13.9 27.8 27.8 27.8                            Magnesium Sulfate                                                                        122.1                                                                              366.2                                                                              30.5 122.09                                                                             61.04                                                                              180.7                                                                              122.09                                                                             180.7                           anhydrate                                                                     Manganese Sulfate H.sub.2 O                                                              10   23.788                                                                             22.5 10   5    22.3 10   22.3                            Molybdenum Trioxide                                                                      --   0.001                                                                              --   --   --   --   --   --                              Molybdic Acid (sodium                                                                    0.25 --   0.062                                                                              0.25 0.125                                                                              0.25 0.25 0.25                            salt) 2H.sub.2 O                                                              Potassium Chloride                                                                       --   65   --   --   --   --   --   --                              Potassium Iodide                                                                         0.75 0.75 0.175                                                                              0.75 0.375                                                                              --   0.75 --                              Potassium Nitrate                                                                        2500 80   625  2500 1250 --   2500 --                              Potassium Phosphate                                                                      --   --   10   --   --   170  --   170                             (monobasic)                                                                   Potassium Sulfate                                                                        --   --   --   --   --   990  --   990                             Sodium Phosphate                                                                         130.5                                                                              16.5 32.62                                                                              130.5                                                                              65.25                                                                              --   130.5                                                                              --                              (monobasic anhydrous)                                                         Sodium Sulfate                                                                           --   200  --   --   --   --   --   --                              Zinc Sulfate 7H.sub.2 O                                                                  2    3    0.5  2    1    8.6  2    8.6                             Myo-Inositol                                                                             100  100  125  100  50   100  100  100                             Nicotinic Acid                                                                           1    --   0.75 1    0.5  1    1    1                               Pyridoxine-HCl                                                                           1    --   0.25 1    0.5  1    1    1                               Thiamine-HCl                                                                             10   5    3.5  10   5    10   10   10                              Glutamine  292.6                                                                              146.4                                                                              --   292.8                                                                              292.8                                                                              1756.8                                                                             --   292.8                           Tryptophan --   --   --   --   --   --   --   --                              Phenylalanine                                                                            --   30   --   --   --   --   --   --                              Lysine     --   20   --   --   --   --   --   --                              Methionine --   --   --   --   --   --   --   --                              Sodium Acetate  10   10   --   --   --   --   --                              Sucrose    10000                                                                              50000                                                                              40000                                                                              10000                                                                              10000                                                                              10000                                                                              10000                                                                              10000                           N.sub.6 -Benzyladenine                                                                   0    2    2    0.002                                                                              0.002                                                                              --        --                              Ascorbic Acid                                                                            50   100  50   100  100  100  100  100                             Picloram   --   --        1.2  2.4  1.2  --   1.2                             Casein hydrolysate                                                                       --   --   500  --   --   --   1000 --                              6-Dimethyltallylamino                                                                    --   --   --   --   --   0.02 --   --                              Purine                                                                        Kinetin    --   --   --   --   --   --   --   0.02                            pH         5.6  5.6  5.6  5.6  5.6  5.6  5.6  5.6                             β-Naphthaleneacetic                                                                 0.931                                                                              10   --   --   --   --   1.862                                                                              --                              Acid                                                                          __________________________________________________________________________

Example 2 Suspension initiation and Growth of Suspended Cells

One gram of callus material was aseptically inoculated into a 125 mlErlenmeyer flask containing 25 ml of liquid medium (Table 1). The flaskwas covered with a silicone foam cap and placed on a gyratory shaker at120 rpm at 24° C. in darkness. Suspension cultures were formed inapproximately 3-10 days. Medium exchanged was initiated by suctionfiltering the flask contents through a buchner funnel containing amiracloth filter and resuspending all the biomass in fresh medium. Oneto two grams of cells were transferred into a 125 ml flask containing 25ml of fresh medium weekly. Typical growth rates and cell densitiesachieved in suspension cultures of representative species are listed inTable 2.

                  TABLE 2                                                         ______________________________________                                        Growth Profile of Taxus Cells                                                          Dry Weight Fresh Weight                                                                             Dry Wt.                                                                             Fresh Wt.                                Species  Doubling Time                                                                            Doubling Time                                                                            Density                                                                             Density                                  ______________________________________                                        T. brevifolia                                                                          2.0 days   3.5 days   20 g/L                                                                              400 g/L                                  T. baccata                                                                             2.0 days   6.0 days   15 g/L                                                                              220 g/L                                  T. chinensis                                                                           2.5 days   4.5 days   20 g/L                                                                              285 g/L                                  T. canadensis       8.5 days   13 g/L                                                                              260 g/L                                  ______________________________________                                    

The increase in biomass (fresh and dry weight) with time for T.chinensis line K-1 was plotted in FIG. 3. The Maximal growth rate wasdetermined by measuring the slope at points of most rapid biomassincrease. Cell cultures of T. chinensis with a maximum doubling time of2.5 days, has a growth rate significantly higher than previouslyreported for Taxus species suspension cultures. Typical Taxus cultureshave doubling times of about 7-12 days.

Culturing cells at high density maximizes the productivity of the cellculture process. Whereas previous cultures of T. brevifolia has a celldensity of less than 1 gram dry weight per liter (Christian et al.,1991), suspension cultures T. chinensis can reach densities up to 8-20grams dry weight per liter after 18 days of growth. Cell viability wasdetermined by staining with a 0.05% fluorescein diacetate (FDA) inacetone (J. M. Widholm, Stain Technol 47:189-94, 1972) followed bycounting the number of green fluorescein cells upon excitation with bluelight in a fluorescence microscope. Cell viability was higher than 90%throughout the growth phase.

Example 3 Viability of Taxus Cells After Preculturing with Mannitol

Six to 7 day old suspension cultures of Taxus cells were harvested andresuspended into fresh growth medium containing 0.16 M mannitol, 0.22 Mmannitol, 0.33 M mannitol or 0.44 M mannitol.

After 3 days of incubation in growth medium with mannitol, cells werecold acclimated at 4° C. for 3 hours. Acclimated cells were harvestedand transferred to 4 ml cryovials containing a cold vitrifying solutionof 40/30 wt % ethylene glycol/sorbitol in media. The vials wereincubated at 4° C. for 3 minutes and frozen by immersion into liquidnitrogen. Vials were maintained in liquid nitrogen for at least 10minutes before use in the thawing experiments.

Vials of frozen cells were removed from liquid nitrogen storage andagitated at 40° C. until the contents are liquefied (1-2 minutes). Theliquefied cells were then washed 5 to 6 times with sterile mediacontaining 1 M sorbitol, 3 times with media containing 0.5 M sorbitolmedia, 3 times with. 0.1 M sorbitol media, and 3 times with sorbitolfree media. Washing was performed by resuspension of cells in washmedia, centrifugation at 50×g for 2 minutes and aspiration of wash mediafrom the cell pellet. Cell viability was determined immediately afterthawing. The summary of multiple experiments is listed below.

                  TABLE 3                                                         ______________________________________                                        Post Thaw Viability of Cells Pretreated with Mannitol                         Concentration                                                                             Viability       Regrowth                                          ______________________________________                                        0.16M       60%             vigorous                                          0.22M       30%             slight                                            0.33M       30%             slight                                            0.44M       20%             slight                                            ______________________________________                                    

Example 4 Viability of Taxus Cells After Preculturing With Sorbitol

Frozen Taxus cells were thawed and suspended into fresh growth mediumcontaining sorbitol at 0.15 M, 0.22 M, 0.33 M, 0.44 M and 0.80 Msorbitol. Cell viability was determined immediately after thawing. Asummary of the results from multiple trials are listed below:

                  TABLE 4                                                         ______________________________________                                        Post Thaw Viability of Cells Pretreated with Sorbitol                         Concentration                                                                             Viability       Regrowth                                          ______________________________________                                        0.15M       20%             none                                              0.22M       40%             vigorous                                          0.33M       30%             vigorous                                          0.44M       20%             vigorous                                          0.80M       20%             slight                                            ______________________________________                                    

Example 5 Viability of Taxus After Preculturing With Sucrose

Six to seven day old cell suspensions in growth medium were harvestedand the cell biomass resuspended in fresh growth medium containing 0.06M, 0.12 M, 0.15 M, 0.29 M and 0.58 M sucrose. Cells were cryopreserved,frozen, thawed and osmotically adjusted accordingly. Cell viability wasdetermined immediately after thawing. A summary of the results frommultiple experiments are listed in Table 5:

                  TABLE 5                                                         ______________________________________                                        Post Thaw Viability of Cells Pretreated with Sucrose                          Concentration                                                                             Viability       Regrowth                                          ______________________________________                                        0.06M       40%             slight                                            0.12M       40%             slight                                            0.15M       40%             slight                                            0.29M       30%             slight                                            0.58M       <15%            slight                                            ______________________________________                                    

Example 6 Effects of Osmotic Agents on the Survival of Taxus Cells

Various osmotic agents in growth medium were evaluated to determinetheir effects on the survival of Taxus species cells precultured withthe agents, after the preculture period and after thawing of thecryoprotected and frozen Taxus cell suspensions. Cells of three-day cellculture suspensions were precultured in growth medium containing variousosmotic agents prior to cryoprotection. Cryoprotected cells were frozenand stored in liquid nitrogen for a minimum of one hour. Viability testswere performed at the end of the preculture period (control) andimmediately after thawing of the cryoprotected and frozen cell.

Cell cultures pretreated with mannitol in growth medium exhibited thehighest percent viability upon thawing after cryoprotection and freezingas compared to viability observed using the other osmotic agents.

                  TABLE 6                                                         ______________________________________                                        Effects of Osmotic Agent on Post Thaw Viability                                              Concentration Survival                                                        (Viability)                                                    Osmotic Agent    Control Frozen                                               ______________________________________                                        Proline          <50%    <15%                                                 Trehalose        50-95%  <15%                                                 Sucrose          50-95%  20-50%                                               Sorbitol         50-95%  30-70%                                               Mannitol         50-95%  40-80%                                               ______________________________________                                    

Example 7 Effect Osmotic Agents and Cryoprotectants on Taxus Viability

Cells of Taxus suspension cultures (KS1A) were harvested and preculturedwith various osmotic agents in the medium. Osmotic agents tested includetrehalose, proline, sorbitol (0.15 M-0.8 M), sucrose (2-20%) andmannitol (0.16 M). Viability was evaluated for each cell suspension atthe end of the preculture period and immediately after thawing. Regrowthwas evaluated after post-thaw osmotic adjustment. The vitrificationsolutions used were ethylene glycol/sorbitol/pectin and ethyleneglycol/sorbitol at 40/30 weight percent in culture medium. The resultsare summarized in Table 7. The highest percent viability and mostrigorous regrowth were observed when mannitol was used for preculturingand ethylene glycol/sorbitol was used as the cryoprotectants in thevitrification solution.

                  TABLE 7                                                         ______________________________________                                        Effects of Osmotic Agents and Cryoprotectants of Post Thaw Viability          Osmotic                     Recovery                                          Agent  Viability                                                                              Cryoprotectants                                                                           Viability                                                                            Growth                                     ______________________________________                                        Trehalose                                                                            50-95%   Ethylene/glycol/                                                                          <15%   none                                       and             Sorbitol/pectin                                               Proline                                                                       Sorbitol                                                                             50-95%   Ethylene glycol/                                                                           30-70%                                                                              slight to vigorous                         0.15 M-         Sorbitol                                                      0.8 M                                                                         Sucrose                                                                              50-95%   Ethylene glycol/                                                                          <10-40%                                                                              none to vigorous                           2%-20%          Sorbitol                                                      Mannitol                                                                             75-95%   Etbylene glycol/                                                                           40-80%                                                                              slight to vigorous                                         Sorbitol                                                      ______________________________________                                    

Example 8 The Effect of Preculture Length on Survival

Taxus cells were harvested from cell culture and the biomass resuspendedin growth medium containing mannitol at a concentration of 3% for oneday or three-days at room temperature. Loaded cells were incubated at 4°C. for 3 hours and transferred to 4 ml cryovials containing coldvitrifying solution comprising 40/30 weight percent ethyleneglycol/sorbitol in culture medium. The vials were incubated at 4° C. forthree minutes and frozen by liquid nitrogen immersion. Cells containedin the vials were maintained in liquid nitrogen for at least 10 minutes.

Cryopreserved cells were thawed and their viability was determined byFDA and tryptan blue staining procedures. Cells precultured withmannitol for three days exhibited significantly higher post-thawavailability than cells which were not precultured in medium containingmannitol.

                  TABLE 8                                                         ______________________________________                                        Effects of Preculture Time on Viability                                                      Survival                                                       Days of Preculture                                                                             Control 3% Mannitol                                          ______________________________________                                        1                5-10%   50%                                                  3                5-15%   40-80%                                               ______________________________________                                    

Example 9 Effect of Ethylene Glycol/Sorbitol on Thawed Taxus CellViability

Six to seven day cell suspensions of Taxus species cell line KS1A werepretreated with 3% mannitol for three days at room temperature. Loadedcells were acclimated to the cold by incubating the flasks at 4° C. for3 hours. Cold acclimated cells were transferred to 4 ml cryovials andcold vitrification solution was added to each and mixed. Aftervitrification at 4° C. for three minutes, cells were frozen by liquidnitrogen immersion. Vials were maintained in liquid nitrogen for atleast 10 minutes.

Cryopreserved cells were thawed by transferring vials from liquidnitrogen and agitated in a 40° C. water bath for 1-2 minutes. Post-thawviability was determined by FDA staining assay.

Ten trials evaluating cell suspensions of Taxus species cell line KS1Awere performed with different concentrations of ethyleneglycol/sorbitol. The results are summarized in Table 9. Cell suspensionsfrozen in the vitrification solution containing ethylene glycol/sorbitolat a concentration of 40/30 wt %, exhibited the highest post-thawpercent viability as well as the most vigorous regrowth as compared tocells vitrified using other concentrations of ethylene glycol/sorbitol.

                  TABLE 9                                                         ______________________________________                                        Effects of Vitrification Solution of Recovery                                 Ethylene Glycol/Sorbitol                                                                     Post-Thaw Viability                                                                          Regrowth                                        ______________________________________                                        50%/30%        20%            slight                                          45%/35%        25%            slight                                          40%/40%        25%            slight                                          40%/30%        60%            vigorous                                        38%/32%        40%            vigorous                                        36%/34%        35%            moderate                                        35%/35%        35%            moderate                                        30%/40%        40%            slight                                          30%/40%        40%            slight                                          20%/50%        25%            none                                            ______________________________________                                    

Example 10 Effects of Cryoprotectants of Taxus Cells Survival After-196° C. Storage

Cultured Taxus cells were harvested and resuspended in fresh growthmedium containing 3% mannitol for 3 days. Cells were cold acclimated for3 hours and transferred to cryovials containing vitrification solution.Cell suspensions were frozen by liquid nitrogen immersion. Liquidnitrogen frozen cells were thawed by agitating the cryovials in a 40° C.water bath for 1-2 minutes. Cells frozen with ethylene glycol as acryoprotectant has the highest viability.

                  TABLE 10                                                        ______________________________________                                        Effects of Cryoprotectants on Viability                                       Cryoprotectant                                                                            Concentration   Viability                                         ______________________________________                                        DMSO         5%-30%         ≦15%                                       Propylene Glycol                                                                          15%              0                                                Glycerol    20%-30%          0                                                PEG-8000    10%              0                                                Ethylene Glycol                                                                           20%-50%          25%-80%                                          ______________________________________                                    

Example 11 Viability of Taxus Cells as a Function of Biomass toVitrifying Solution

Six to seven day old cell suspension of taxus species cell line KS1Awere harvested and resuspended in fresh medium containing 3% mannitoland incubated for three days at room temperature. Following coldacclimation at 4° C. for 3 hours, cells were transferred to 4 mlcryovials containing 40/30 wt % ethylene glycol/sorbitol in culturemedium. Vials were incubated at 4° C. for 3 minutes and frozen by liquidnitrogen immersion. Vials were maintained in liquid nitrogen for atleast 10 minutes before thawing.

The highest percent viability was observed when the cell biomass/vitrifying solution quantity was 167 mg/ml. Acceptable viability wasalso observed when the cell biomass/vitrifying solution ratio was 143,200 and 250 mg/ml.

                  TABLE 11                                                        ______________________________________                                        Effects of Cell Biomass on Viability                                          Cell Biomass/                                                                 Vitrifying Solution       Viability                                           ______________________________________                                        143         mg/ml         60%                                                 167         mg/ml         80%                                                 200         mg/ml         45%                                                 250         mg/ml         45%                                                 300         mg/ml         ≦10%                                         400         mg/ml         ≦10%                                         500         mg/ml         ≦5%                                          1,000       mg/ml         ≦5%                                          ______________________________________                                    

Example 12 Effects of Different Method Steps on Taxus Cell Viability

Different method steps were evaluated to determine the steps which wouldresult in the highest percent post-thaw viability. Cells werecryopreserved with and without cryoprotectants, with and without osmoticpretreatments, with and without cold treatment, and with and withoutvitrification.

In the first trial, six to seven day old Taxus cells cultures werefrozen with and without cryoprotectants. In the second trial, cells werefrozen with and without a 40/30, weight percent, ethyleneglycol/sorbitol vitrification solution treatment. In the third trial,cells were vitrified and frozen with and without a pretreatmentcomprising a three day incubation in 3% mannitol growth media. In thefourth trial, cells were pretreated and vitrified and frozen with orwithout cold acclimation.

For each trial, viability tests were performed immediately afterthawing. Cells precultured with growth medium containing 3% mannitol for3 days at room temperature, followed by a 2-4 hour cold treatment priorto cryoprotection, exhibited the highest percent viability. Suitableviability was also observed in cells precultured for 3 days in mediumcontaining 3% mannitol and subjected to cryoprotection without previouscold treatment, and in cells preculture in growth medium for 3 days andprecultured in media containing mannitol for 24 hours followed by a 2 to4 hour cold treatment prior to cryoprotection.

                  TABLE 12                                                        ______________________________________                                        Viability of Cells Recovered from Liquid Nitrogen                             Treatment      Viability     Regrowth                                         ______________________________________                                        Cells in medium                                                                              0             none                                             Direct plunging into                                                          liquid nitrogen                                                               Cells in medium                                                                              ≦10    none                                             cryoprotection                                                                liquid nitrogen                                                               Cells in medium                                                                              40-60         slight                                           precultured 3 days                                                            in 3% mannitol                                                                cryoprotection                                                                liquid nitrogen                                                               Cells in medium                                                                              40-80         vigorous                                         Precultured 3 days                                                            in 3% mannitol                                                                2-4 hour cold                                                                 treatment                                                                     cryoprotection                                                                liquid nitrogen                                                               Cells in growth                                                                              40-60         vigorous                                         medium for 3 days                                                             preculture in osmotic                                                         media for 24 hours                                                            3% mannitol                                                                   2-4 hour cold                                                                 treatment                                                                     cryoprotection                                                                liquid nitrogen                                                               ______________________________________                                    

Cells were again tested for viability tests using the indicated steps,performed according to the methods described herein.

                  TABLE 13                                                        ______________________________________                                        Viability of Cells Recovered from Liquid Nitrogen                             Treatment    Viability      Regrowth                                          ______________________________________                                        Freeze Dried 20%            none                                              Liquid nitrogen                                                               Freeze Dried 30-60%         moderate                                          Vitrification                                                                 Liquid nitrogen                                                               Freeze Dried 20-40%         slight                                            Preculture                                                                    in sorbitol                                                                   Liquid nitrogen                                                               Freeze Dried 30-60%         moderate                                          Preculture                                                                    in sorbitol                                                                   Vitrification                                                                 Liquid nitrogen                                                               Freeze Dried 20-40%         slight                                            Loading                                                                       Vitrification                                                                 Liquid nitrogen                                                               Freeze Dried 30-50%         good                                              Loading                                                                       Vitrification                                                                 Liquid nitrogen                                                               Preculture   40-60%         good                                              in sorbitol                                                                   Freeze Dried                                                                  Vitrification                                                                 Liquid nitrogen                                                               Preculture   40-60%         good                                              in mannitol                                                                   Freeze Dried                                                                  Vitrification                                                                 Liquid nitrogen                                                               Preculture   40-60%         good                                              in sucrose                                                                    Freeze Dried                                                                  Vitrification                                                                 Liquid nitrogen                                                               ______________________________________                                    

Example 13 Cell Viability and Growth Before and After Cryopreservation

The following Taxus species cell lines were evaluated to determine cellviability and regrowth after cryopreservation:

KS1A; KEIR; 647; 1224; 12-6;12-14; and 12-20

Six to seven day old cell suspensions of each cell line were harvestedand the biomass resuspended in fresh growth medium containing 3%mannitol. Cells were incubated for 3 days at room temperature andthereafter the loaded cells suspensions were incubated at 4° C. for from3 hours. Cold acclimated cells were transferred to 4 ml cryovialscontaining a cold vitrification solution of ethylene glycol/sorbitol40/30 wt %. The vitrification solution and cells were gently mixed andthe vials were incubated at 40° C. for 3 minutes. Thereafter, the cellsuspensions were frozen by liquid nitrogen immersion for at least 10minutes.

After freezing, the cells were thawed by transferring the frozen vialsfrom liquid nitrogen to a 40° C. water bath for 1-2 minutes. Post-thawcell viability was determined by FDA staining assay. Cells were washed5-6 times with cold sterile 1 M sorbitol media and resuspended in fresh1 M in medium.

The cell suspensions free from toxic cryoprotectants were then eachseparately filtered using a buchner funnel and Whatmann 541 filter paperunder sterile conditions. For each cell suspension, the filter withcells was layered on semisolid growth medium containing 0.5 M sorbitoland equilibrated for 30 minutes at room temperature. Paper containingcells was transferred to solid growth medium containing 0.1 M sorbitoland incubated for 24 hours. The paper with cells was transferred tosemisolid growth medium without sorbitol and incubated for 24 hours atroom temperature. The filter containing cells was then again transferredto fresh semisolid growth medium without sorbitol and incubated at roomtemperature for an additional 24 hours. Callus cell growth on thesemisolid nutrient media was evident at about 2 to 3 weeks. Thereafter,cell suspensions in liquid growth medium were initiated from the callus.

As can be seen from Table 14 set forth below, all of the cell linesevaluated exhibited acceptable post-thaw viability and recovery growth.

                  TABLE 14                                                        ______________________________________                                        Effects of Preculture Conditions on Viability                                 Cell  Preculture         Cryo-   Post-Thaw                                                                            Recovery                              Line  Condition Viability                                                                              Protectants                                                                           Viability                                                                            Growth                                ______________________________________                                        KS1A  3% mannitol                                                                             ≧95%                                                                            ethylene                                                                              40-80% vigorous                                    3 days             glycol/                                                                       sorbitol                                                                      40/30 wt %                                           KEIR  3% mannitol                                                                             ≧95%                                                                            ethylene                                                                              30-60% slight                                      3 days             glycol/                                                                       sorbitol                                                                      40/30 wt %                                           647   3% mannitol                                                                             ≧95%                                                                            ethylene                                                                              30-60% slight                                      3 days             glycol/                                                                       sorbitol                                                                      40/30 wt %                                           1224  3% mannitol                                                                             ≧95%                                                                            ethylene                                                                              40-60% vigorous                                    3 days             glycol/                                                                       sorbitol                                                                      40/30 wt %                                           12-6  3% mannitol                                                                             ≧95%                                                                            ethylene                                                                              40%    vigorous                                    3 days             glycol/                                                                       sorbitol                                                                      40/30 wt %                                           12-14 3% mannitol                                                                             ≧95%                                                                            ethylene                                                                              30%    vigorous                                    3 days             glycol/                                                                       sorbitol                                                                      40/30 wt %                                           1220  3% maniiitol                                                                            ≧95%                                                                            ethylene                                                                              35%    vigorous                                    3 days             glycol/                                                                       sorbitol                                                                      40/30 wt %                                           ______________________________________                                    

Example 14 Growth and Product Formation of Taxus Cells UponCryopreservation

Six to seven day cell suspensions of Taxus cell line KS1A werecryopreserved and thawed. The cells were precultured with 3% mannitol ingrowth medium for 3 days and 40/30 wt % ethylene glycol/sorbitol wasused as a cryoprotectants. Growth doubling time and product formationwere evaluated before and after freezing and thawing. Product yield wasmonitored after 5 days of growth in suspension. Nuclear DNA content inthe cells was monitored by flow cytometry and found to be about 22.7pg/nuclei before and 22.9 pg/nuclei after cryopreservation.Cryopreservation did not affect product production.

                  TABLE 15                                                        ______________________________________                                        Growth and Product Formation of Taxus before and after LN.sub.2               Preculture                                                                            Cryo-     (Doubling Product Formation (mg/L)                          Treatments                                                                            Protectant                                                                              Time)     Taxol                                                                              Baccatin                                                                             10-DAB                                ______________________________________                                        3% mannitol                                                                           ethylene  7 (5-7)   0.2  1.1    0.4                                           glycol/                                                                       sorbitol/                                                                     (40/30 wt %)                                                                            7 (5-7)   0.2  1.1    0.4                                   ______________________________________                                    

Example 15 Growth and Product Formation in Taxus Cells AfterCryopreservation

Taxus species cell lines were cryopreserved and subsequently thawed andsubjected to post-thaw osmotic adjustment. Growth and product formationwere determined after the establishment of cell suspensions in liquidculture. Growth reflects the average doubling time of cell suspensionsin days after they were established in growth medium. Product formationwas determined after 14 days of growth in suspension. Results are listedin Table 16.

                  TABLE 16                                                        ______________________________________                                        Taxol Production of Cells Recovered from Cryopreservation                              (Average   Production                                                Cell     doubling time                                                                            (mg/L-14 days)                                            Line     in days)   Taxol     Baccatin                                                                             10-DAB                                   ______________________________________                                        Keir     4          10.5      10.6   3.4                                      KS1A     5.5        22.6      7.4    2.3                                      1224     5.5        10.8      73     13                                       SS3-184  7          --         --    --                                       SS12-6   5.8        25        51     6.7                                      SS12-19  6          9.2       31     4.7                                      SS12-20  5.5        10        24     4.4                                      SS12-79  5          2.4       9.6    1.9                                      SS12-99  8.5        --        --     --                                       SS12-103 6          19.3      68.5   14.3                                     647      5.5        --        --     --                                       ______________________________________                                    

FIG. 4 illustrate chromatograms of the extracellular fraction at day 20from Taxus species cell line 1224 where (A) represents the control cellsuspension which was not cryopreserved and (B) represents the cellsuspension regenerated after cryopreservation, freezing, storage for sixmonths, and thawing and post-thaw osmotic adjustment. Peak 1 is10-deacetylbaccatin; peak 2 is 9-dihydrobaccation III; peak 3 isbaccatin III; peak 4 is 9-dihydro-13-acetylbaccatin III; peak 5 istaxol; peak 6 is 2-benzoyl-2-deacetylbaccatin and peak 7 is2-benzoyl-2-deacetyl-1-hydrozybaccatin I.

FIG. 5 illustrate chromatograms of the extracellular fraction at day 20from Taxus species cell line 203 where (A) represents the control cellsuspension Which was not cryopreserved and (B) represents the cellsuspension regenerated after cryopreservation, freezing, storage forthree months, and thawing and post-thaw osmotic adjustment. Peak 1 is10-deacetylbaccatin; peak 2 is 9-dihydrobaccation III; peak 3 isbaccatin III; peak 4 is 9-dihydro-13-acetylbaccatin III; peak 5 is taxoland peak 6 is 2-benzoyl-2-deacetylbaccatin. As can be seen from FIGS. 4and 5, the product formation profile is substantially the same in thecontrol cell suspension and the regenerated cell suspension.

Example 16 Genetic Stability of Cryopreserved and Non-CryopreservedCells

Cell lines were established from a single Taxus chinensis var. mairertree. One of these established cell lines was cultured, cryopreservedfor one year, and thawed. Genetic analysis was performed on cells fromthe original tree and on the cryopreserved cells to determine ifcryopreservation had affected genetic stability of the cells. Briefly 10μg of total DNA from each cell line was and treated with a four foldover digestion of restriction endonuclease and size fractionated byagarose gel electrophoresis. The size fractionated DNA was transferredto a nitrocellulose solid support and hybridized to a radio labelednucleic acid probe, Jeffrey's 33.6 minisatellite probe. Thishypervariable region probe shows different banding patterns from the DNAof 4 separate trees in lanes 1-4 of FIG. 6. In contrast, the initialisolate (lane A), cells cultured for 1 year (lane B), and cellscryopreserved for 1 year (lane C) was identical genetically from cellsisolated from the same tree one year later (lanes D and E).Cryopreservation did not result in any mutation detectable by thisanalysis.

Example 17 Stability of Cryopreserved Taxus Cells

To determine if the length of cryopreservation has any effect on geneticstability, Taxus cell lines 1224 was frozen for one hour to 6 months andanalyzed for their genetic stability. DNA was extracted from viablecultures grown thawed and re-established from these cryopreserved cells.A 3.1 Kb polymorphic region of the genome containing nuclear ribosomalcoding and non-coding DNA was amplified by polymerase chain reaction anddigested with endonuclease DpnII. The digested DNA was sorted by sizeusing gel electrophoresis and visualized after ethidium bromidestaining. The results of the analysis are shown in FIG. 7. The originalcell line and a noncryopreserved cell line were analyzed in lanes C andD respectively. Two unrelated cell lines established from unrelatedtrees show a different digestion pattern. In contrast, no geneticmutation was detected in cells cryopreserved for one hour (lane E), oneday (lane F), one week (lane G), one month (lane H) or 6 months (lane Iand J). The banding pattern of these cryopreserved and non-cryopreservedcells were all identical (lanes C to J).

Other embodiments and uses of the invention will be apparent to thoseskilled in the art from consideration of the specification and practiceof the invention disclosed herein. All U.S. patents cited herein arehereby specifically incorporated by reference in their entirety. Thespecification and examples should be considered exemplary only with thetrue scope and spirit of the invention indicated by the followingclaims.

We claim:
 1. A method for cryopreserving a plant cell comprising thesteps of: pretreating the plant cell with a cryoprotective agent and astabilizer, wherein said stabilizer is an antioxidant or a radicalscavenger; and in any order, acclimating the pretreated plant cell to areduced temperature; loading the plant cell with a loading agent; andvitrifying the plant cell with a vitrification solution; and freezingthe vitrified plant cell to a cryopreservation temperature.
 2. Themethod of claim 1 wherein the plant cell is a gymnosperm or anangiosperm.
 3. The method of claim 2 wherein the gymnosperm is a speciesof Abies, Cypressus, Ginkgo, Juniperus, Picea, Pinus, Pseudotsuga,Sequoia, Taxus, Tsuga or Zamia.
 4. The method of claim 3 wherein theTaxus species is T. baccata, T. brevifolia, T. canadensis, T chinensis,T. cuspidata, T. floridana, T. globosa, T. media, T. nucifera or T.wallichiana.
 5. The method of claim 2 wherein the angiosperm is amonocotyledon plant cell or a dicotyledon plant cell.
 6. The method ofclaim 5 wherein the monocotyledon plant cell is selected from the groupconsisting of species of the genus Avena, Cocos, Dioscorea, Hordeum,Musa, Oryza, Saccharum, Sorghum, Triticum and Zea.
 7. The method ofclaim 5 wherein the dicotyledon plant cell is selected from the groupconsisting of species of the genus Achyrocline, Atropa, Brassica,Berberis, Capsicum, Catharanthus, Conospermum, Datura, Daucus,Digitalis, Echinacea, Eschscholtzia, Glycine, Gossypium, Hyoscyamus,Lycopersicum, Malus, Medicago, Nicotiana, Panax, Pisum, Rauvolfia, Ruta,Solanum and Trichosanthes.
 8. The method of claim 1 wherein the plantcell is obtained from new growth needles, bark, leaves, stem, root,rhizome, callus cells, protoplasts, cell suspensions, meristems, seedsor embryos.
 9. The method of claim 1 wherein pretreatment involvesculturing said plant cell in medium containing said loading agent andsaid stabilizer for between about 1 hour to about 7 days at about roomtemperature.
 10. The method of claim 1 wherein the loading agent is asugar, an amino acid or a combination thereof.
 11. The method of claim10 wherein the sugar comprises one or more sugars selected from thegroup consisting of fructose, glucose, maltose, mannitol, sorbitol,sucrose, trehalose and derivatives thereof.
 12. The method of claim 10wherein the amino acid is proline.
 13. The method of claim 1 wherein theloading agent is employed as an aqueous solution at a concentration ofbetween about 0.05 M to about 0.8 M.
 14. The method of claim 1 whereinthe loading agent is employed as an aqueous solution at a concentrationof between about 1% to about 10% by weight.
 15. The method of claim 1wherein the stabilizer is selected from the group consisting of reducedglutathione, tetramethylurea, tetramethylthiourea, dimethylformamide,mercaptopropionyl glycine, mercaptoethylamine, selenomethionine,thiourea, dimercaptopropanol, ascorbic acid, cysteine, sodiumdiethyldithiocarbomate, sodium thiosulfate, silver thiosulfate,propylgallate, spermine, spermidine and combinations and derivativesthereof.
 16. The method of claim 1 wherein the stabilizer is employed asan aqueous solution at a concentration of between about 1 μM to about 1mM.
 17. The method of claim 1 wherein the reduced temperature is betweenabout 1° C. to about 15° C.
 18. The method of claim 1 wherein loadingcomprises incubating said plant cell in a solution comprising betweenabout 0.5% to about 10%, by weight, of a vitrifying agent.
 19. Themethod of claim 1 wherein the vitrification solution comprises avitrifying agent selected from the group consisting of DMSO, propyleneglycol, glycerol, polyethylene glycol, ethylene glycol, butanediol,formamide, propanediol, sorbitol, mannitol and mixtures thereof.
 20. Themethod of claim 1 wherein the vitrification solution comprises betweenabout 20% to about 60%, by weight, of a vitrifying agent.
 21. The methodof claim 1 wherein the vitrification solution is added to the plant cellto establish a biomass to solution ratio of about 250 mg/ml or less. 22.The method of claim 1 wherein the loading agent is a vitrifying agent.23. The method of claim 1 wherein loading and vitrifying are performedsubstantially simultaneously.
 24. The method of claim 1 wherein thecryopreservative temperature is less than about -70° C.
 25. The methodof claim 1 wherein the cryopreservative temperature is between about-70° C. to about -200° C.
 26. The method of claim 1 further comprisingthe step of storing the cryopreserved cell at said cryopreservationtemperature for a period of time of greater than one month.
 27. Themethod of claim 26 wherein the period of time is greater than one year.28. The method of claim 1 wherein pretreating is preformedsimultaneously with loading and the loading agent and the cryoprotectiveagent are the same.
 29. The method of claim 1 wherein pretreating ispreformed simultaneously with vitrifying and the vitrification soulutioncontains the cryoprotective agent.